Previous attempts to quantify or determine an enzyme or its activity involved one of at least four general methods. Enzyme activity can be measured in vivo or in vitro by the ability of an enzyme, generally purified, to hydrolyze its natural substrate to yield a measurable product. Secondly, enzyme activity can also be measured by the ability of an enzyme to hydrolyze a synthetic substrate. Thirdly, an enzyme can be measured by an immunoassay, typically by a radioimmunoassay using tagged anti-enzyme antibody or tagged enzyme. Lastly, an enzyme can also be measured by the use of enzyme inhibitors that are simple organic reagents, not proteinaceous.
All of these methods for quantifying enzymes suffer from the lack of specificity of the tagged binding partner of the enzyme, e.g. natural or synthetic substrate, anti-enzyme antibody or inhibitor, for the enzyme. This lack of specificity is a serious problem with bioassays of enzymes in a biological fluid, wherein the assayed enzyme is often present in extremely small quantities, e.g. urinary kallikrein has a concentration normally in the range of about 150 nanograms of kallikrein/ml in urine normally in the range of 50-300 micrograms of protein/ml. In a biological sample, e.g. urine or plasma, the likelihood that the ligand interacts substantially with undesired proteins is high. For example, diisopropylfluorophosphate (DFP) is known to react with a specific serine hydroxyl group in acetyl-cholinesterase [Means, G. E. et al., Chemical Modification of Proteins Holden-Day, Inc. (1971), p. 26], yet it will react with a wide variety of enzymes for example serine proteases present in urine.
Bioassays measure biological effects but tend to be tedious, may require expensive animal preparations, are imprecise, and typically rely on hydrolysis of unknown and variable concentrations of native substrates. Radioimmunoassays often provide the greatest sensitivity coupled with good specificity. However, immunoassays generally fail to distinguish active enzyme from inactive enzyme, enzyme precursor(s) or enzyme bound to an inhibitor. Furthermore, radioimmunoassay requires labelled enzyme or labelled antibody, neither of which is inexpensive to prepare and store.
The present invention provides two sources of specificity for an assay of any protease (also known as proteinase) for which naturally-occurring proteinaceous inhibitors are available, whereas previous methods generally provided only one source of specificity. By binding a protease with a tagged inhibitor, i.e. a tagged proteinaceous inhibitor of natural origin, to form protease-inhibitor complexes, then adding anti-protease antibodies, separating protease-inhibitor complexes bound to antibody from the tagged inhibitor, and determining the amount of tag in either the bound or free fraction, the sensitivity of the radioimmunoassay is maintained and the specificity for the active form of a protease is improved. It will be understood that one source of specificity in the protease assay of this invention is the particular binding characteristics of the anti-protease, the other is the particular binding characteristics of the tagged proteinaceous inhibitor.
Furthermore, the present invention is completely different from the "sandwich" assay. The sandwich assay is a method of determining the concentration of an antigen, e.g. a protease, by binding the antigen to its corresponding antibody in solid-phase, then adding tagged purified antibody [Kabat, E. A. Structural Concepts in Immunology and Immunochemistry 2nd Ed. Holt, Rinehart and Winston (1976) p. 75; Salmon, S. E. et al. J. Immunol. 103, 129 (1969)]. The essential differences are that (1) the sandwich assay requires the use of a tagged purified antibody, preferably .sup.125 I-affinity purified antibody, an expensive and very unstable reagent to make in the laboratory, (2) the sandwich assay does not use a tagged proteinaceous inhibitor of proteases, and (3) the sandwich assay is not an activity assay for an enzyme, particularly a protease.
U.S. Pat. No. 4,134,792 describes a specific binding assay with an enzyme modulator as a labeling substance. The modulator is an inhibitor or allosteric effector and appears to be understood as any simple organic reagent, not a proteinaceous inhibitor. Furthermore, this patent describes an assay substantially different from that of the present invention because the inhibitor is measured by determining the change in the activity of the enzyme, whereas the present invention determines the enzyme by measuring the amount of tagged inhibitor bound to the enzyme.
Several other patents describe methods of isolating naturally occurring proteinaceous inhibitors of proteases or conversely, methods of isolating enzymes that are known to have affinity for such proteinaceous inhibitors of proteases. See, inter alia U.S. Pat. Nos. 3,181,997; 3,558,773; 3,630,841; 3,834,990; 4,030,977. Since these patents describe purification schemes and do not employ a tag on the inhibitor or the enzyme, it is apparent that the novel assay procedure of the present invention is a clearly different and novel use of naturally occurring proteinaceous inhibitors of proteases.
The present invention retains the well-known sensitivity of a radioimmunoassay, but substantially avoids the undesired measurement of denatured enzyme. The present invention avoids the pitfalls typically found in the performance of a radioimmunoassay or a sandwich assay, i.e. preparation of labelled enzyme or of labelled antibody. Enzyme inhibitors, particularly proteinaceaous inhibitors, tend to be highly stable and require less expense and time for isolation than the proteases which they bind. Hence it is generally easier to prepare labelled inhibitors than labelled protease. It is well known that labelled, highly purified antibodies are unstable; they tend to break down or denature rapidly even during storage below -60.degree. C.
A wide variety of plasma proteases have naturally occurring proteinaceous inhibitors. Many of the plasma proteins are of substantial importance in the clinical evaluation of disease. They are, for example, involved in blood coagulation, blood clot dissolution, formation and destruction of peptide hormones, complement cascade activation [Laskowski et al. Ann. Rev. Biochem. 49, 593 (1980)], and other important natural functions. The present invention encompasses a new and better method of assaying these proteases by measuring their capacity to bind to both a tagged proteinaceous inhibitor and to a specific anti-protease antibody or other appropriately specific binding partner of the protease.